1. Field of the Invention
This invention relates to a method for immobilizing the enzyme on a water-insoluble carrier and to an immobilized enzyme produced thereby.
More particularly, this invention relates to a method for immobilizing an enzyme on a water-insoluble amino group-containing carrier such as silica gel, porous glass or the like having amino groups introduced thereinto, by use of a polyfunctional crosslinking agent in the presence of a phenolic carboxylic acid having one or more hydroxyl groups such as tannic acid or the like or in the presence of a phenolic carboxylic acid having one or more hydroxyl groups and chitosan. In addition, the present invention is also directed to the immobilized enzymes obtained by said method.
According to this invention, an enzyme can be stably immobilized on a water-insoluble carrier while retaining its high activity, thereby producing an immobilized enzyme which permits continuous enzymatic reactions for a long period of time. Through the use of the immobilized enzyme of this invention, the purity of a product obtained by enzymatic reaction can be improved, the amount of enzyme used can be reduced, and the enzymatic reaction vessel required for the enzymatic process can be made more compacted.
2. Related Art Statement
Enzymatic reactions are widely used in industrial production processes of medicines, foods etc. Such enzymatic reactions have heretofore been carried out in solutions prepared by dissolving an enzyme in an aqueous solution of substrate. However, such methods require not only very complicated procedures, for example, the steps of supplying fresh enzyme while keeping the reaction conditions constant and of recovering the enzyme after the reaction, but also very troublesome procedures of separating and purifying the reaction product. In order to remove these defects, continuous enzymatic reactions directed to the use of an enzyme immobilized on a water-insoluble carrier have been investigated in recent years.
Along this line, the main methods for immobilizing enzymes include: known, for example, (1) the carrier covalent binding method, (2) the physical adsorption method or the ionic binding method, (3) the crosslinking method and (4) the entrapping method.
Among these immobilization methods, the carrier covalent binding method of (1) permits production of an immobilized enzyme in which the binding power between the carrier and the enzyme is relatively strong, but is disadvantageous in that the determination of optimal conditions for the covalent binding reaction is difficult, and that the production of an immobilized enzyme having high activity is also generally difficult. The physical adsorption method or ionic binding method of (2) permits immobilization of an enzyme by a simple procedure under mild conditions and hence the production of an immobilized enzyme having relatively high activity, but is disadvantageous in that since the binding power between the carrier and the enzyme is weak, the enzyme tends to be released from the carrier. The crosslinking method of (3) is disadvantageous in that since the crosslinking reaction is carried out under relatively severe conditions, only an immobilized enzyme having low activity can be obtained. The entrapping method of (4) is generally advantageous in that an immobilized enzyme can be produced easily at low cost, but is disadvantageous not only in that when a water-soluble polymer such as polyacrylamide is crosslinked and then gelatinized for entrapping an enzyme, the enzymic activity is unavoidably lowered, but also in that the enzyme is liable to be released from the gel formed.
As the method for immobilizing the enzyme according to the carrier covalent binding method of (1), there may be exemplified, for example, a method which comprises forming Schiff bases or peptide bonds between the functional groups (--NH.sub.2, --COOH, etc.) on the surface of a carrier and the --NH.sub.2, --COOH, etc. groups present in the enzyme molecules by use of a polyfunctional crosslinking agent or a condensing agent. A method comprising activating an amino group-containing carrier by use of a crosslinking agent such as glutaraldehyde, and then immobilizing glucoamylase thereon is described in Starch/Stark 33 (1981) Nr. 2, S.52-55. Further, immobilization of glucoamylase on aminated silica gel by use of glutaraldehyde is described in Enzyme Microb. Technol., 1982, Vol. 4, Mar. pp. 89-92. However, the half life of the enzymic activity obtained by these methods is short, so that the period of time of stable saccharification produced by these immobilized enzymes is also short. Therefore, continuous saccharification using these immobilized enzymes cannot be carried out for a long period of time unless there is employed, for example, a method comprising placing a large number of reaction tanks in parallel and using them one after another. In this case, complicated apparatuses and procedures are required and the enzyme should be frequently renewed.
As another carrier covalent binding method, there is a method which comprises immersing a molded product of swollen chitin in an enzyme solution, then carrying out glutaraldehyde treatment, thereby producing an immobilized enzyme (Japanese Patent Application Kokai (Laid-Open) No. 111686/86). However in the case of such a method, it is also difficult to obtain a stably immobilized enzyme retaining high activity.
As described above, the immobilization processes of the prior art, in particular, the carrier covalent binding method, have many disadvantageous, for example, insufficient consideration has been given to the affinity of the carrier surface for the enzyme, determination of conditions for covalent binding reaction is complicated, and it is difficult to obtain an immobilized enzyme having high activity.
On the other hand, methods for producing an immobilized enzyme by the crosslinking method include, for example, a method comprising the steps of coagulating a cell enzyme by use of a macromolecule coagulant such as chitosan, and then carrying out a crosslinking reaction through the use of glutaraldehyde or the like to immobilize the cell enzyme (Japanese Patent Application Kokai (Laid-Open) No. 120182/77); a method comprising using tannin together with a coagulant such as polyethyleneimine, and then immobilizing an enzyme by use of a crosslinking agent such as glutaraldehyde (Japanese Patent Application Kokai (Laid-Open) No. 110190/82); and a method comprising the steps of using polyethyleneimine as a coagulant, carrying out a crosslinking reaction in the presence of chitosan through the use of glutaraldehyde, thereby immobilizing an enzyme (Japanese Patent Application Kokai (Laid-Open) No. 58072/85). These methods use chitosan or tannin, but in all of them, immobilized enzymes are obtained by the crosslinking methods. Thus, they are different from the carrier covalent binding method in which an enzyme is immobilized on a water-soluble carrier.